Francisella tularensis causes a potentially life-threatening disease called tularemia. This pathogen can be aerosolized, survive in the environment and has an infectious dose of as few as 10 organisms. This has led the CDC and NIAID to classify this organism as a category A select agent and a potential agent of biological warfare. F. tularensis is a gram-negative coccobacillus that can infect a wide variety of vertebrate and invertebrate hosts. In mammalian hosts it primarily lives and replicates inside macrophages. The mechanisms by which the organism colonizes, invades and survives inside the host cell are mostly unknown. We have chosen to focus this proposal on microbial adherence since for many pathogens this is a key step in pathogenicity. Our overall hypothesis is that adhesions are critical virulence factors that are required for pathogenicity. A search of the genomic sequences of the virulent strain F. tularensis Schu4 has identified several loci with homology to genes involved in type IV pili structure and biogenesis. In other bacteria, which have a similar type of pilus, these pili have been shown to be critical virulence factors. This has led to our specific hypothesis: Type IV pili mediate attachment to host cell macrophages and help to facilitate host cell invasion. In this proposal we will focus on identifying and characterizing the gene(s) encoding pilin, the structural subunit of type IV pili, and investigating the role of this pilus in adherence, invasion and twitching motility. Specifically we will verify the presence of type IV pili by microscopy and immune sera. We will isolate pili from F. tularensis and sequence the pilin subunit by mass spectrometry analysis. We will purify pili for antibody production. To explore the diversity of the pilin subunit we will also clone and sequence the pilin encoding genes from the live vaccine strain (LVS) and additional clinical isolates of F. tularensis. We will develop an adherence and invasion assay using bacteria that express the gene encoding the green fluorescent protein. We will use pilin-specific antibody to try to block adherence and/or invasion. We will construct a pilin minus strain of F. tularensis and test its ability to adhere to and invade host cells and exhibit twitching motility. Understanding the basic mechanisms of pathogenicity will lead to a greater understanding of why this organism is such an effective pathogen, and the identification of new targets for therapies.